What blood tests are recommended for diagnosing Polycystic Ovary Syndrome (PCOS)?

PCOS Diagnostic Bloodwork

Measure total testosterone (TT) and free testosterone (FT) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as your first-line laboratory tests for diagnosing biochemical hyperandrogenism in PCOS. 1, 2

First-Line Androgen Testing

The 2025 International PCOS Guidelines explicitly recommend TT and FT as mandatory first-line tests because they provide the best diagnostic accuracy. 1, 2

• Total testosterone demonstrates pooled sensitivity of 74% and specificity of 86% for PCOS diagnosis 1
• Free testosterone shows superior sensitivity of 89% with specificity of 83% 1, 2
• LC-MS/MS is the required assay method, showing superior specificity (92%) compared to direct immunoassays (78%) 1, 2, 3
• If LC-MS/MS is unavailable, use Free Androgen Index (FAI) as an alternative, which has sensitivity of 78% and specificity of 85% 1, 2
• Calculated free testosterone (cFT) should be assessed by equilibrium dialysis, ammonium sulfate precipitation, or calculated using FAI 1, 2

Second-Line Androgen Testing (If TT/FT Normal)

If total or free testosterone are not elevated but clinical suspicion remains high, proceed to measure:

• Androstenedione (A4): sensitivity 75%, specificity 71% 1, 2
• DHEAS (dehydroepiandrosterone sulfate): sensitivity 75%, specificity 67% 1, 2

Important caveat: A4 and DHEAS have significantly poorer specificity than TT/FT and should only be used as adjunctive tests, not primary diagnostic markers 1, 2

Mandatory Exclusion Tests

These tests rule out conditions that mimic PCOS:

• Thyroid-stimulating hormone (TSH) to exclude thyroid disease as a cause of menstrual irregularity 2, 3
• Prolactin (morning resting serum levels) to exclude hyperprolactinemia 2, 3
• 17-hydroxyprogesterone to exclude non-classical congenital adrenal hyperplasia 2, 3

Metabolic Screening (Mandatory for All PCOS Patients)

All women with PCOS require metabolic screening regardless of BMI, as insulin resistance occurs independently of body weight. 3

• Two-hour oral glucose tolerance test (75g glucose load) to screen for glucose intolerance and type 2 diabetes 2, 3
• Fasting lipid panel: total cholesterol, LDL, HDL, and triglycerides 2, 3
• Body mass index (BMI) calculation 2, 3
• Waist-hip ratio to identify central obesity (WHR >0.9 indicates truncal obesity) 2

Optional Supportive Tests

These tests can provide additional diagnostic information but are not required:

• LH and FSH measured between cycle days 3-6, with LH/FSH ratio >2 suggesting PCOS, though this is abnormal in only 35-44% of PCOS cases, making it a poor standalone marker 2, 4
• Mid-luteal progesterone (<6 nmol/L indicates anovulation) to confirm ovulatory dysfunction 2
• Anti-Müllerian hormone (AMH) ≥35 pmol/L shows 92% sensitivity and 97% specificity, but should NOT replace ultrasound or serve as a standalone diagnostic test due to lack of assay standardization 2, 3

Critical Pitfalls to Avoid

Do not rely on LH/FSH ratio as a primary diagnostic criterion - it has poor sensitivity (abnormal in only 35-44% of PCOS cases) and should be abandoned as a biochemical criterion 2, 4

Do not assume normal testosterone excludes PCOS - total testosterone is abnormal in only 70% of women with confirmed PCOS, meaning 30% have normal levels despite having the condition 2

Do not use direct immunoassays for testosterone measurement - they have significantly lower specificity (78%) compared to LC-MS/MS (92%), leading to false positives 1, 2

Do not skip metabolic screening in lean women - insulin resistance and metabolic dysfunction occur independently of BMI in PCOS 3

Diagnostic Algorithm Summary

1. First: Measure TT and FT using LC-MS/MS (or FAI if LC-MS/MS unavailable) 1, 2
2. Second: If TT/FT normal but clinical suspicion high, measure A4 and DHEAS 1, 2
3. Simultaneously: Measure TSH, prolactin, and 17-hydroxyprogesterone to exclude mimicking conditions 2, 3
4. Always: Perform metabolic screening with OGTT and lipid panel regardless of BMI 2, 3

Remember that PCOS can be diagnosed based solely on clinical hyperandrogenism plus irregular menstrual cycles without any abnormal laboratory values, per Rotterdam criteria. 2 The absence of biochemical hyperandrogenism does not exclude PCOS when clinical features and ultrasound findings are present.